Skip Navigation

Cardiovascular Research 2004 62(3):468-480; doi:10.1016/j.cardiores.2004.02.006
© 2004 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Janczewski, A. M
Right arrow Articles by McTiernan, C. F
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Janczewski, A. M
Right arrow Articles by McTiernan, C. F
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2004, European Society of Cardiology

Phospholamban gene ablation improves calcium transients but not cardiac function in a heart failure model

Andrzej M Janczewskia, Maliha Zahida, Bonnie H Lemstera, Carol S Fryea, Gregory Gibsona, Yoshihiro Higuchib, Evangelia G Kraniasc, Arthur M Feldmanb and Charles F McTiernan*,a

aCardiovascular Institute, University of Pittsburgh, 200 Lothrop Street, 1744.1 BST, Pittsburgh, PA 15213, USA
bDepartment of Medicine, Thomas Jefferson University, USA
cDepartment of Pharmacology and Cell Biophysics, University of Cincinnati, USA

* Corresponding author. Tel.: +1-412-648-3016; fax: +1-412-383-8857. Email address: mctiernanc{at}msx.upmc.edu

Received 7 August 2003; revised 6 February 2004; accepted 9 February 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Decreased amplitude and slower kinetics of cardiomyocyte intracellular calcium (Cai2+) transients may underlie the diminished cardiac function observed in heart failure. These alterations occur in humans and animals with heart failure, including the TNF1.6 mouse model, in which heart failure arises from cardiac-specific overexpression of tumor necrosis factor {alpha} (TNF{alpha}). Objective: Since ablation of phospholamban expression (PLBKO) removes inhibition of the sarcoplasmic reticulum (SR) Ca2+ pump, enhances SR Ca2+ uptake and increases contractility, we assessed whether ablation of phospholamban expression could improve cardiac function, limit remodeling, and improve survival in the TNF1.6 model of heart failure. Methods: We bred PLBKO with TNF1.6 mice and characterized the progeny for survival, cardiac function (echocardiography), cardiac remodeling (hypertrophy, dilation, fibrosis), and Cai2+ transients and contractile function of isolated cardiomyocytes. Results: PLB ablation did not improve survival, cardiac function, or limit cardiac chamber dilation and hypertrophy in TNF1.6 mice (TKO mice). However, contractile function and Cai2+ transients (amplitude and kinetics) of isolated TKO cardiomyocytes were markedly enhanced. This discordance between unimproved cardiac function, and enhanced Cai2+ cycling and cardiomyocyte contractile parameters may arise from a continued overexpression of collagen and decreased expression of gap junction proteins (connexin 43) in response to chronic TNF{alpha} stimulation. Conclusions: Enhancement of intrinsic cardiomyocyte Cai2+ cycling and contractile function may not be sufficient to overcome several parallel pathophysiologic processes present in the failing heart.

KEYWORDS Calcium pump; Contractile function; Cytokines; Heart failure; Transgenic animal models; Mice

Abbreviations: PLB, phospholamban • SERCA, sarcoplasmic reticulum Ca2+ATPase • UTR, untranslated region • WT, wild type • KO, PLB knockout • Tg, TNF1.6 transgenic • TKO, TNF1.6 lacking PLB • FS%, percent fractional shortening • EDD, end diastolic dimension • ESD, end systolic dimension • GAPDH, glyceraldehyde phosphate dehydrogenase • NCX, sodium calcium exchanger


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Sarcoplasmic reticulum (SR) calcium (Ca2+) cycling plays a significant role in defining cardiac contractility, with alterations in Cai2+ cycling observed in both animal models of heart failure and in the human failing myocardium. Key components of SR Ca2+ uptake are the SR Ca2+ pump (SR Ca2+ ATPase, SERCA2) and phospholamban (PLB). PLB inhibits SERCA2 [1] by increasing the Ca2+ dependence of catalytic activity [2], leading to a slower decrease in cytosolic Ca2+ and prolonging the time of muscle relaxation [3]. This inhibition is removed by phosphorylation of PLB, allowing faster rates of SR Ca2+ uptake, muscle relaxation, and enhancement of contractility through increased SR Ca2+ loading [4,5]. While diminished Ca2+i transient amplitude and slowed rates of SR Ca2+ uptake and transient decline are observed in isolated muscles and cells from failing human hearts [6,7], the underlying biochemical mechanisms remain uncertain. However, altered expression of transcripts encoding SR Ca2+ transport proteins (such as SERCA2 and PLB) [8–11], altered expression level and phosphorylation status of multiple SR proteins [12–14], and functional differences in SR Ca2+ uptake and release [12,13] are observed in the failing human heart and direct speculation as to the role of the SR in heart failure.

Inhibition of SERCA2 by PLB can also be limited by decreasing the relative expression of PLB to SERCA2. Ablation of the PLB gene (PLB-KO mice) or transgenic overexpression of PLB demonstrates the relationship between the PLB/SERCA2 ratio and SR Ca2+ uptake and relaxation parameters [15,16]. Furthermore, enhanced expression of PLB or SERCA2 from recombinant adenoviral constructs demonstrates the PLB/SERCA2 ratio as a key determinant of contractile function [17,18]. Therefore, strategies to enhance cardiac function in heart failure may seek to reduce the relative expression of PLB to SERCA2 [19].

TNF1.6 mice develop a well-characterized heart failure as a consequence of cardiac-specific overexpression of tumor necrosis factor alpha (TNF-{alpha}). This is demonstrated by ventricular hypertrophy and dilatation, interstitial fibrosis, attenuation of adrenergic responsiveness, reduction in developed pressures and ejection fraction, and overt congestive heart failure, with a six-month mortality of 25% [20–22]. Furthermore, hearts and isolated cardiomyocytes from TNF1.6 mice demonstrate Ca2+i transients with diminished amplitudes and slowed kinetics relative to that observed in wild type mice [23,24], resembling observations from human failing hearts [6,7]. Interestingly, male TNF1.6 mice have an earlier onset of severe heart failure relative to female TNF1.6, although older females will also eventually develop heart failure [21,22]. In this report, we investigated whether chronic absence of PLB expression could enhance Cai2+ transient kinetic parameters and attenuate the heart failure and remodeling that develops in TNF1.6 mice. Thus, we bred PLB-KO to TNF1.6 mice and assessed cardiac function, and measured the contractile and Cai2+ transient parameters in isolated cardiomyocytes in progeny with four different genotypes; wild type (WT), PLB-KO (KO), TNF1.6 transgenic (Tg), and TNF1.6 lacking PLB (TKO).


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Animal models
PLB-KO mice [15] were backcrossed into the FVB background for at least six generations; TNF1.6 mice [20] were generated and maintained in the FVB strain. Breeding TNF1.6 to PLB-KO mice created heterozygous mice (F1 generation). PCR screening methods were used to identify the TNF-{alpha} transgene status [20]. Identification of PLB gene status utilized forward primers corresponding to 3' UTR sequences of the mouse PLB gene (5'-cttggtcactactgtcagaac-3') and the neomycin cassette (5'-tcctcgtgctttacggtatc-3') and a reverse primer corresponding to 3'UTR sequences of the mouse PLB gene (5'-tgtgggttgcaaagttaggc-3'). Breeding F1 male heterozygous PLB-KO to F1 female transgene positive/heterozygous knock-out created 12 classes in the F2 generation: M or F with six genotypes; wild type (WT), heterozygous knockout (He), homozygous knockout (KO), TNF1.6 transgene positive (Tg), transgene positive with heterozygous knockout (THe) and transgene positive with homozygous knockout (TKO). Littermates of the desired genotypes were selected with male and female mice analyzed separately due to the sex-dependent differences in the severity of heart failure in TNF1.6 mice [21,22,24]. The investigation conforms with the Guide for the Care and Use of Laboratory Animals (US National Institutes of Health Publication No. 85-23, revised 1996).

2.2. Cardiomyocyte studies
Cardiomyocyte isolation, and measurement of Cai2+ and cell shortening have been described previously [24]. Single left ventricular myocytes were obtained by enzymatic digestion. For measurements of Cai2+ indexed by the fura-2 ratio, cells were loaded with 5 µM fura-2 AM for 30 min. Mechanical measurements were performed in cardiomyocytes not loaded with fura-2. Cardiomyocytes were studied in a temperature-controlled chamber (25 °C) on the stage of an inverted microscope (Nikon Diaphot 300), perfused with a modified Tyrode solution [24], and electrically stimulated at 0.5 Hz. To achieve more ‘physiologic’ conditions, additional experiments were performed at 36 °C and 5 Hz. Steady-state contractions or Cai2+ transients (5–10 each) were averaged for each cell and used to analyze the measured parameters.

2.3. Echocardiography
At 7–12 weeks of age, mice were anesthetized (2.5% Avertin i.p.) and echocardiography was performed [21]. Two-dimensional targeted M-mode imaging was obtained from the short-axis immediately below the level of the mitral valve. M-mode tracings were recorded and measurements of LV end-diastolic and end-systolic diameter were made by using the leading-edge method for at least three cardiac cycles on the M-mode tracings. LVFS (%) was calculated as previously reported [21]. Measurement processing was performed with the operator blinded to the mouse genotypes. After echocardiography, mice were euthanized and processed for cardiomyocyte isolation or collection of tissues for biochemical studies.

2.4. Survival
Survival was assessed using the Kaplan–Meier survival analysis (SPSS software).

2.5. Cardiac hypertrophy and histology
Hearts were recovered, rinsed in cold phosphate buffered saline (PBS), biventricular weights recorded, and tissues flash frozen in liquid nitrogen. Cardiac hypertrophy was determined by the biventricle (mg) to body weight (g) ratio. Tissue sections were stained with hemotoxylin and eosin as previous reported [20].

2.6. Western blots
Protein extracts were prepared in RIPA buffer and Western blot analyses performed [24]. Additional antibodies included anti-sodium–calcium exchanger (NCX, 1:1000 Swant {pi}11–13), and anti-calsequestrin (1:2500 Affinity Bioreagents PA1-913). Chemiluminescent signals were normalized to that obtained from anti-glyceraldehyde phosphate dehydrogenase (GAPDH, 1:1000, Research Diagnostics TRK5G4-6C5) and were in turn normalized to the mean of the male and female WT samples combined, arbitrarily set at 100%.

2.7. Immunohistochemistry
Excised cardiac tissues were rinsed in ice cold PBS, and processed for immunofluorescent analyses [22], with a rabbit anti-collagen I antibody (1:500, Chemicon #AB765), rabbit anti-connexin antibody (1:250, Zymed #71-0700), and a rabbit anti-desmoplakin antibody (1:1000, Serotec AHP 320). Sections were stained with phalloidin (Molecular Probes A22287 [GenBank] ) to identify filamentous actin. Fluorescent secondary antibodies (Jackson ImmunoResearch Laboratories) included goat anti-rabbit IgG conjugated with CY3 (1:1000), and goat anti-rabbit conjugated with Alexa 488 (1:500). Slides were viewed at 40X with a confocal microscope (Olympus FV500). Images were collected with a cooled CCD camera (Optronic Magnifier) at 24-bit gray depth and assembled (Adobe Photoshop). Five images were taken for each specimen stained with phalloidin and anti-collagen I. For each animal, the fractional area occupied by collagen (collagen area divided by the sum area occupied by collagen and phalloidin) was calculated as previously described [22], and the mean per group determined.

2.8. Statistical analysis
Results are reported as mean±standard error of the mean (S.E.M.). Comparisons between more than one group were performed using ANOVA with Student–Newman–Keuls post-hoc tests. Differences were considered significant at p<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Calcium transients in isolated cardiomyocytes
Intracellular calcium (Cai2+) transients (Fig. 1 and Table 1) were analyzed to assess whether PLB gene ablation normalized the depressed Cai2+ transients observed in TNF1.6 mice [23,24]. Due to sex-dependent differences in the severity of heart failure at a given age [21,22,24], males and females were analyzed separately. Relative to WT male cardiomyocytes, Cai2+ transients in KO male mice showed the expected increased amplitude and faster kinetics of maximum rate of Cai2+ rise, decline, and time to 50% Cai2+ transient decline, whereas Tg male cardiomyocytes showed significantly lower amplitudes and slower kinetics (Table 1). Diastolic fura-2 fluorescence ratio, an index of diastolic CaI2+, was not statistically different among the experimental groups (Table 1). Male TKO cardiomyocytes demonstrated a significantly increased Cai2+ transient amplitude, faster Cai2+ transient kinetics, and shorter T50 when compared to either male WT or Tg. However, the Cai2+ transient amplitude and kinetics remained lower and slower relative to those observed in male KO cardiomyocytes.


Figure 1
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 PLB gene ablation enhances Cai2+ transients in isolated cardiomyocytes. (a) Representative Cai2+ transient traces shown from male WT, KO, Tg, or TKO cardiomyocytes loaded with fura-2 and electrically stimulated to contract at 0.5 Hz in the presence of 2 mM extracellular Ca2+([Ca2+]o) at 25 °C. Original recordings show intracellular (Cai2+) transients indexed by fura-2 ratio 380/360 nm (bottom) and their first derivative (top).

 

View this table:
[in this window]
[in a new window]

  		Table 1 Intracellular Ca2+ activity and contractions in isolated cardiomyocytes

 
Female WT and KO cardiomyocytes showed no significant difference in Cai2+ transient amplitude or kinetics when compared to male cardiomyocytes of the same genotype (Fig. 1 and Table 1). Cai2+ transients in female Tg cardiomyocytes showed significant differences from WT cardiomyocytes only in the longer T50. Similar to male cardiomyocytes, female TKO cardiomyocytes showed enhanced amplitude and faster Cai2+ transient kinetics relative to female WT or Tg cardiomyocytes. However, as for males, the Cai2+ transient amplitudes and kinetics in TKO female cardiomyocytes were diminished compared to female KO cardiomyocytes.

3.2. Contractile properties of isolated cardiomyocytes
To determine whether the increased amplitude and kinetics of the Cai2+ transient were reflected in the cardiomyocyte contractile function, we measured the extent of shortening ({Delta}L(%)) and maximal rates of contraction and relaxation in female WT, KO, Tg, and TKO cardiomyocytes (Fig. 2 and Table 1). As previously reported under the same experimental conditions (25 °C, stimulation at 0.5 Hz) [24], the contractile parameters were not significantly different between female Tg and WT cardiomyocytes while, as expected [25], KO cardiomyocytes showed significantly increased {Delta}L(%) and maximal rates of contraction and relaxation (Table 1). Similarly, female TKO showed a significantly greater {Delta}L(%) and faster rates of contraction and relaxation compared to either WT or Tg cardiomyocytes, although these values were significantly less than those that were observed in KO cardiomyocytes.


Figure 2
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 PLB gene ablation enhances contractile parameters in isolated cardiomyocytes. (a) Representative contraction traces from female WT, KO, Tg, and TKO cardiomyocytes. Cells were not loaded with fura-2 but were otherwise stimulated to contract as described in Fig. 5 legend. Original tracings show positive and negative deflections of dL/dt (top) which reflect maximal rate of cell shortening and relaxation of unloaded cell shortening, respectively, and FS% (bottom) represents percentage change from diastolic length.

 
These contractile activity studies were performed under commonly utilized conditions (~25 °C and 0.5-Hz stimulation rate). However, these differ from the conditions of temperature and contraction rate at which the heart normally operates. To address this possible limitation, studies were performed to examine the contractile function of male WT, Tg and TgKO myocytes at "near physiological" conditions with respect to temperature and pacing rate (36 °C and 5 Hz). Under these conditions, the {Delta}L(%) in WT cell averaged 49% more compared to Tg, while the {Delta}L(%) in TKO cells was 85% larger compared to Tg and 24% larger compared to WT cells, although the latter difference did not reach statistical significance (Fig. 3). The maximal rate of shortening was significantly reduced (by 61%) in Tg vs. WT and significantly increased, by 31% and 116%, in TKO vs. WT and Tg myocytes, respectively. The maximal rate of relaxation was significantly reduced (by 32%) in Tg vs. WT and significantly increased, by 76% and 157%, in TKO vs. WT and Tg myocytes, respectively. Thus, at 36 °C and 5-Hz stimulation rate, cardiomyocytes from male Tg mice also displayed a decreased extent of shortening and slower kinetics than male WT cardiomyocytes, whereas TKO mice displayed an enhanced extent of shortening and kinetics of contraction and relaxation.


Figure 3
View larger version (28K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 PLB gene ablation rescues contractile parameters in isolated cardiomyocytes at 36 °C and 5-Hz stimulation. Original tracings (averaged for 10 steady-state beats) showing dL/dt (top) and {Delta}L(%) (middle) in representative myocytes isolated from male WT, Tg and TKO mice. Bottom, mean values±S.E. from these experiments. Analyses on 21 cells/2 hearts (WT), 17 cells/2 hearts (Tg), 9 cells/1 heart (TKO). p<0.05, * vs. WT, {ddagger} vs. Tg.

 
3.3. Echocardiographic studies
To assess whether improvements in contractile properties observed in TKO cardiomyocytes were reflected in the contractile properties of intact hearts, we assessed cardiac function by echocardiography. Typical M-mode echocardiographic images and data are presented in Fig. 4 and Table 2. WT and PLB-KO mice demonstrated fractional shortening percentage (FS%), and left ventricular end systolic (LVESD) and end diastolic chamber dimensions (LVEDD) similar to previous reports comparing basal FS% between WT and PLB-KO mice [26]. However, both Tg and TKO mice demonstrated a significant reduction in FS% and enhanced LVEDD and LVESD relative to either WT or KO mice of the same sex, as well as a decreased relative ventricular wall thickness (h/r). Furthermore, LVEDD and LVESD were significantly greater in male TKO relative to male Tg mice.


Figure 4
View larger version (113K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 PLB gene ablation does not improve systolic function or reduce ventricular dilation in TNF1.6 mice. M-mode echocardiographic images of male wild type (WT), PLB-KO (KO), TNF1.6 transgenic (Tg) and TNF1.6 transgenic with PLB gene ablation (TKO). Bars indicate end-diastolic and end-systolic points.

 

View this table:
[in this window]
[in a new window]

  		Table 2 Echocardiographic analyses

 
3.4. Cardiac hypertrophy and histology
There were no significant differences in cardiac hypertrophy among non-transgenic mice (WT, He, KO) of the same sex regardless of PLB gene number (Fig. 5A). In both sexes, Tg mice showed significant cardiac hypertrophy relative to any non-transgenic mice (WT or KO). In both sexes, TKO mice demonstrated significantly greater hypertrophy relative to Tg mice of the same sex. Neither Tg nor TKO mice displayed any obvious cardiomyocyte disarray, but did display myocardial infiltrates (Fig. 5B) as previously reported for mice with cardiac-specific overexpression of TNF-{alpha} [20].


Figure 5
View larger version (58K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 PLB gene ablation does not limit cardiac hypertrophy or alter histology appearance in TNF1.6 mice. (A) He, non-transgenic with heterozygous PLB gene ablation; THe, transgenic with heterozygous PLB gene ablation; other abbreviations as in Fig. 1 legend. N=4–18 per group. Values are mean±S.E.M. p<0.05, * vs. WT, He, KO; {dagger} vs. Tg, THe (same sex). NS, not significant. (B) Representative H+E images from female WT, KO, Tg, and TKO mice.

 
3.5. Survival analyses
In both males and females, there were no significant differences in survival among non-transgenic mice (WT, He, KO) of the same sex regardless of PLB gene number (Fig. 6). In males (Fig. 6A), Tg mice showed a significant reduction in 90-day survival compared to WT, similar to that previously reported [21]. In males, both THe and TKO mice showed a significantly reduced survival relative to Tg mice. In females (Fig. 6B), no significant difference in survival was noted over the first 90 days between Tg and WT, similar to that previously reported [21]. A trend towards reduced survival of female THe and TKO did not reach statistical significance.


Figure 6
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 PLB gene ablation does not improve survival in TNF1.6 mice. Kaplan–Meier survival analysis of TNF1.6 mice with various PLB gene copy numbers. N=11–46 per group. p<0.05, * vs. WT; {dagger} vs. KO; {ddagger} vs. Tg; NS, not significant.

 
3.6. Western blot analyses
Western blot analyses were performed to assess the potential contribution of altered expression of key Ca2+ regulating proteins to contractile dysfunction. Previous studies had observed no differences in the expression of PLB and SERCA2 proteins between WT and TNF1.6 mice [24]. Current analysis confirmed and extended these observations with no significant differences observed in the expression of SERCA2, calsequestrin, or the NCX regardless of transgene or PLB gene status (Fig 7; quantitative data not shown), and the expected lack of PLB expression in PLB-KO mice.


Figure 7
View larger version (48K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Western blot analysis of Ca2+ regulatory protein expression. Representative Chemiluminescent image of Western blot analyses. PLB, phospholamban; NCX, Na+–Ca2+ exchanger; SERCA, SR Ca2+ ATPase; CALQ, calsequestrin; GAPDH, glyceraldehyde phosphate dehydrogenase.

 
3.7. Cardiac collagen deposition
The discordance between enhanced contractile properties of isolated cardiomyocytes and deficient global cardiac function with reduced survival suggests altered efficiency or coordination of force production by cardiomyocytes within the intact TKO heart relative to that of the isolated cardiomyocyte. To assess whether PLB gene ablation may alter the extracellullar matrix remodeling and fibrosis observed in TNF1.6 mice [22], immunohistochemical staining was performed for collagen type I. No significant difference in the deposition of collagen type I was observed between female WT and KO ventricular tissues (Fig. 8A, B). However, a substantial deposition of collagen was observed in both Tg and TKO mice (Fig. 8C, D), with the extent of collagen expression (%fractional area) higher in TKO relative to Tg mice (Fig. 8E). Similar results were also observed in male mice (data not shown).


Figure 8
View larger version (85K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8 PLB gene ablation does not alter extent of collagen deposition in TNF1.6 mice. Representative frozen sections from (A) WT, (B) KO, (C) Tg, and (D) TKO female mice stained with anti-collagen I antibody (green) and phalloidin (red). (E) Deposition of collagen (percent fractional area) is greater in TKO than WT, KO, or Tg mice. Number of animals examined (all female) in parentheses. p<0.05, * vs. all other groups.

 
3.8. Cardiomyocyte connexin 43 expression
Alterations in cardiomyocyte function in the intact heart could also arise from changes in gap junction-mediated intercellular communication and coordination of electrical depolarization [27]. Immunohistochemical analyses were performed with anti-desmoplakin to assess intercalated disk distribution and anti-connexin 43, which defines myocardial gap junction properties [27]. While a similar distribution and intensity of connexin 43 expression was observed in WT, KO, and Tg hearts, TKO hearts demonstrated a markedly less intense expression of connexin 43 in cardiomyocyte gap junctions (Fig. 8). Since the distribution of desmoplakin appeared equivalent among the four groups, the reduction in connexin 43 staining appeared to arise from a decreased expression of connexin 43 rather than a decrease in density of intercalated disks (Fig. 9).


Figure 9
View larger version (97K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 9 Decreased connexin 43 expression in TKO cardiomyocytes. Representative confocal microscopic images from frozen sections of (A) WT, (B) KO, (C) Tg, and (D) TKO female mice stained with anti-desmoplakin (red; left), anti-connexin 43 (green; center), and phalloidin (blue with desmoplakin and connexin 43 overlay; right).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Numerous studies have demonstrated that the amplitude and kinetics of the Ca2+i transient are depressed in the human failing heart, in large part due to impaired SR Ca2+ cycling [6,7,12,13]. These alterations produce corresponding changes in Ca2+-dependent myofilament activation and impaired systolic and diastolic function. Alterations in Ca2+ cycling may also modify hypertrophy signal transduction pathways [28,29] and activity of transcription regulatory factors [30] which may mediate further modifications of cardiac structure and function in heart failure. The SR Ca2+ pump (SERCA2) and its endogenous regulator PLB have been extensively studied for their role in the regulation of Ca2+ cycling (see review in Refs. [31,32]). Alterations in the expression, phosphorylation, and/or function of these SR proteins may participate in the pathophysiology of human heart failure [8–14,32]. Since alteration of the PLB/SERCA2 expression ratio can modify SR Ca2+i transient amplitude and kinetics, and contractile function [16–18], it has been proposed that decreasing the PLB to SERCA2 ratio could enhance Cai2+ transients and therefore improve contractile function of the failing heart [19].

One murine model (the TNF1.6 mouse) that recapitulates many aspects of human heart failure arises from cardiac-specific transgenic overexpression of TNF-{alpha} [20–24]. While other carefully characterized murine models of hypertrophy and/or heart failure arise from genetically manipulated expression of single sarcomeric, SR, cytoskeletal, or signal transduction proteins intrinsic to cardiomyocyte function [33–37], the TNF1.6 model utilizes overexpression of an extrinsic and perhaps more ‘upstream’ signal (TNF-{alpha}) that is a proposed soluble mediator of heart failure pathophysiology [38]. While it must be acknowledged that use of the {alpha}-MHC promoter to achieve life-long, unregulated transgenic gene expression could lead to physiologic adaptations or responses different from an adult onset overexpression of the gene in question, we have extensively characterized the TNF1.6 model and have observed structural and functional remodeling consistent with classical markers of heart failure [20–24]. Thus, in this study, TNF1.6 mice were chosen to assess whether a genetic ‘complementation’ method to remove PLB expression could limit cardiac dysfunction through enhancement of SR Ca2+ cycling in the continued presence of a stimulus (TNF-{alpha}) that drives the development of heart failure.

The current study yielded three important observations. First, PLB gene ablation fully restored Cai2+ transient parameters and contractile activity of isolated cardiomyocytes in heart failure (TNF1.6) mice. Furthermore, the impaired contractile function in male Tg myocytes and its rescue by PLB ablation (TKO) were sustained when studied under "near physiological" conditions (with respect to temperature and stimulation rate), consistent with similar studies performed at lower temperature and stimulation rate. Second, the enhancement of cardiomyocyte Cai2+ transient and contractile function occurred independently of and without improvements in global cardiac function in vivo, hypertrophy, ventricular dilation, or survival. Indeed some worsening of these measures was observed. Finally, we observed persistent alterations in extracellular matrix and components regulating intercellular communication in the myocardium of these heart failure mice, which may prevent improvements in cardiac function despite the improvement of Cai2+ handling and the cardiomyocyte contractile function.

Divergent responses have been observed in prior studies assessing whether PLB gene ablation could improve global cardiac structure and/or function in genetic models of cardiac hypertrophy or heart failure [33–37,39,40]. Such studies were successful only in cardiomyopathies associated with a reduction of both the amplitude and kinetics of the Cai2+ transient [37,39], i.e. akin to human heart failure [6,7], but not in those associated solely with a slower decay of the Cai2+ transient [35,40]. In the current study, regardless of whether the myocytes displayed decreased Cai2+ transient amplitude (males) or not (females), the rescue of the Cai2+ transient amplitude and kinetics by PLB ablation failed to improve cardiac structure or function. These findings may reflect the complexity of cardiomyopathies (including human heart failure) consequent to simultaneous activation of multiple, independent pathways (which may occur after chronic overexpression of soluble ‘upstream’ factors, e.g. TNF-{alpha}) vs. those arising from genetic manipulations of specific proteins that are ‘downstream’ of signal transduction pathways.

Not only did PLB gene ablation fail to improve cardiac function and limit remodeling, cardiac hypertrophy was greater in TKO relative to Tg in both male and female mice, and dilation was increased (males) or trended towards an increase (females) in TKO mice relative to TG mice. Several mechanisms may underlie this exacerbation of remodeling and cardiac dysfunction. For example, PLB-KO mice display a small but significant increase in mean arterial pressure [41], which could amplify the hypertrophy and remodeling observed in TNF1.6 mice. Additionally, intracellular Ca2+ is a second messenger involved in the signaling pathways cascade activated by stretch [42], which may be regulated by the amplitude and duration of Cai2+ signals [43]. Thus, in the setting of heart failure consequent to TNF-{alpha} overexpression, deleterious effects of PLB ablation on cardiac remodeling and function in vivo may partly stem from increased amplitude and/or kinetics of the Cai2+ transient. Finally, PLB ablation produces chronic enhancement of SR Ca2+ cycling and increased basal metabolic demands [44]. Indeed PLB-KO mice show an increased susceptibility to ischemic injury with a greater net utilization of ATP [45]. As enhanced TNF-{alpha} expression is a significant component of ischemic injury to the heart [46], the combination of increased TNF-{alpha} and PLB ablation may be particularly injurious to the myocardium.

Interestingly, unlike PLB-KO mice, humans that express a truncated, non-functional PLB (Leu-39 Stop, a functional PLB knockout) spontaneously develop cardiomyopathy and heart failure in early adulthood [47]. A variable severity of clinical presentation in humans heterozygous for the Leu-39 Stop allele suggests other environmental stimuli or genetic factors modifying the consequence of reduced or absent functional PLB. It is interesting to speculate that such factors may be mimicked in PLB-KO mice which over-express TNF-{alpha}. However, the different outcomes between functional PLB ablation in mice and humans may also arise from fundamental differences in cardiac physiology and Ca2+ cycling in these two species [48].

Of further interest is the gender-dependent difference in severity of heart failure induced in Tg and TKO mice. An earlier onset and a greater severity of cardiac dysfunction in male vs. female TNF1.6 mice [21,22,24] may arise from a greater TNF-{alpha}-activated production of ceramide in male hearts [21]. Other animal models also reveal a more severe heart failure/hypertrophy in males relative to females [49,50]. Some of the mechanisms underlying gender-related differences in hypertrophy and failure may arise from altered telomerase activity [51], differential regulation of p38 MAK kinase [52], and effects of sex hormones on translation and/or function of proteins implicated in cardiac structure, contractile function and Ca2+ homeostasis [50,53]. Furthermore, one might speculate that gender-related differences in PLB phosphorylation in male failing human hearts relative to female [13] indicates not only gender-related differences in Ca2+ handling proteins, but in kinases, phosphatases and other (non-PLB) phospho-protein targets that mediate cardiac dysfunction. Thus, gender-related differences in hypertrophy and failure are likely to arise from multiple mechanisms.

While one might hypothesize that enhancement of SR Ca2+ cycling would improve cardiac function in heart failure, the results of this study are consistent with prior observations that increasing SR Ca2+ cycling may not be sufficient to improve cardiac function or survival. For example, phosphodiesterase inhibitors increase SR Ca2+ cycling and contractile performance without heart failure survival benefit [54,55]. Also, transgenic mice with overexpression of the phosphatase calcineurin develop heart failure despite increased SR function and contractile performance of isolated cardiomyocytes [56]. Nonetheless, alterations in SR function can improve cardiac function and diminish heart failure severity. Overexpression of a pseudo-phosphorylated form of PLB improves cardiac function and survival in the cardiomyopathic hamster [57]. Differences between these results and those seeking ‘rescue’ of heart failure through PLB ablation suggest a variance between the disinhibition of SERCA2 through PLB ablation or PLB phosphorylation. Indeed, PLB physically associates with multiple proteins [58–60], potentially generating different effects on cellular processes between PLB ablation and chronic phosphorylation despite similar SERCA2 disinhibition. Furthermore, a life-long absence of PLB expression could lead to physiologic alterations not recapitulated by interventions in adult animals that alter PLB expression or activity subsequent to the onset of heart failure.

Ca2+ transient amplitudes and kinetics are influenced by at least three mechanisms; Ca2+ influx through the voltage-gated Ca2+ channel to induce Ca2+ release, Ca2+ release from the SR through the Ca2+ release channel (ryanodine receptor, or RyR2), and the re-uptake of cytosolic Ca2+ into the SR by SERCA2, regulated by PLB. Due to slower decline of the Cai2+ transient in TNF1.6 cardiomyocytes, we suspect that defects in functional properties of SERCA2/PLB and/or RyR2 underlie these abnormal Cai2+ transients [24]. While this study has examined whether enhanced SR Ca2+ uptake may improve function of the failing heart, the nature of the primary Ca2+ handling defect in TNF1.6 mice remains unknown. Recent studies in other models have argued that hyperphosphorylation of RyR2 increases the sensitivity of the SR Ca2+ release channel to Ca2+-dependent activation, potentially leading to depletion of SR Ca2+ stores through a ‘leaky’ channel, reducing the co-ordinate action of neighboring Ca2+ release channels, and reducing the efficiency of EC coupling [14]. Thus, in addition to Ca2+ uptake into the SR, other possible targets remain to enhance or normalize Ca2+ handling and cardiac function in the failing heart.

A potential limitation of the present studies is that, while both Ca2+i transients and contractions were assessed at 25 °C (Figs. 1 and 2Go and Table 1) only contractions were examined at 36 °C (Fig. 3). However, it has been shown previously that the amplitude and kinetics of the Cai2+ transient are depressed in Tg vs. WT mice at body temperature and 5–12-Hz stimulation rates [23], consistent with the present contractile data. Thus, while it seems likely that the same mechanism, i.e. impaired Cai2+ transient, is responsible for the depressed contractility at body temperature and room temperature, future studies should confirm the interpretation.

Most notably, regardless of PLB expression, the myocardium of TNF1.6 mice is exposed to continued signaling from TNF-{alpha} overexpression. TNF-{alpha}-driven alterations in collagen deposition and extracellular matrix remodeling [22] continue despite the enhancement of intrinsic cardiomyocyte contractile properties through PLB gene ablation. Excessive cardiac collagen deposition is associated with increased stiffness and diminished cardiac function [61] and may contribute to altered electrical coupling and coordination of cardiomyocyte contraction [62,63]. Furthermore, cardiac tissue from TKO mice, other murine models of heart failure [35,56], and failing human hearts [64] all show decreased expression of the gap junction protein connexin 43, which together with the connexin 43 phosphorylation status defines intercellular communication and cardiomyocyte electrical coupling [27]. Indeed the TNF1.6 mouse shows heterogeneity of electrical conduction through the myocardium, and develops Ca2+-dependent arrhythmias [23]. Thus, in the TNF1.6 model, changes in collagen deposition and connexin 43 expression are important determinants of cardiac dysfunction that are neither normalized nor compensated through enhancement of Cai2+ transients by PLB ablation.

The present study reinforces the molecular complexity of the cardiac physiologic changes in heart failure. Clearly great caution must be taken both in utilizing results from any single heart failure model, and when extrapolating results from isolated unloaded cardiomyocytes to those from the intact heart. However, in both the heart failure model utilized in this study, and recent observations in other models [35], addressing one component of heart failure (SR Ca2+ cycling and/or contractile function of the isolated cardiomyocyte) may not be sufficient to fully rescue the failing myocardium. Indeed the continued exposure to cytokines or hormonal factors which drive the development of heart failure likely leads to a plethora of physiologic changes which are independent of SR Ca2+ cycling and intrinsic cardiomyocyte function, and which may serve to maintain the heart failure phenotype.


   Acknowledgements
 
This work was supported in part by the Bill Hillgrove Fellowship (A.J., Y.H., University of Pittsburgh Medical Center), and NIH grants HL 26057, HL 64018 and HL 52318 (E.G.K.).


   Notes
 
Time for primary review 26 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Inui M, Chamberlain B.K, Saito A, Fleischer S. The nature of the modulation of Ca2+ transport as studied by reconstitution of cardiac sarcoplasmic reticulum. J. Biol. Chem. (1986) 261:1794–1800.[Abstract/Free Full Text]
  2. Tada M, Kirchberger M.A, Repke D.I, Katz A.M. The stimulation of calcium transport in cardiac sarcoplasmic reticulum by adenosine 3':5'-monophosphate-dependent protein kinase. J. Biol. Chem. (1974) 249:6174–6180.[Abstract/Free Full Text]
  3. Lindemann J.P, Jones L.R, Hathaway D.R, Henry B.G, Watanabe A.M. Beta-adrenergic stimulation of phospholamban phosphorylation and Ca2+-ATPase activity in guinea pig ventricles. J. Biol. Chem. (1983) 258:464–471.[Free Full Text]
  4. Wegener A.D, Simmerman H.K, Lindemann J.P, Jones L.R. Phospholamban phosphorylation in intact ventricles. Phosphorylation of serine 16 and threonine 17 in response to beta-adrenergic stimulation. J. Biol. Chem. (1989) 264:11468–11474.[Abstract/Free Full Text]
  5. Chu G, Lester J.W, Young K.B, Luo W, Zhai J, Kranias E.G. A single site (Ser16) phosphorylation in phospholamban is sufficient in mediating its maximal cardiac responses to beta-agonists. J. Biol. Chem. (2000) 275:38938–38943.[Abstract/Free Full Text]
  6. Gwathmey J.K, Copelas L, MacKinnon R, et al. Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure. Circ. Res. (1987) 61:70–76.[Abstract/Free Full Text]
  7. Beuckelmann D.J, Nabauer M, Erdmann E. Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circulation (1992) 85:1046–1055.[Abstract/Free Full Text]
  8. Mercadier J.J, Lompre A.M, Duc P, et al. Altered sarcoplasmic reticulum Ca2(+)-ATPase gene expression in the human ventricle during end-stage heart failure. J. Clin. Invest. (1990) 85:305–309.[Web of Science][Medline]
  9. Feldman A.M, Ray P.E, Silan C.M, Mercer J.A, Minobe W, Bristow M.R. Selective gene expression in failing human heart. Quantification of steady-state levels of messenger RNA in endomyocardial biopsies using the polymerase chain reaction. Circulation (1991) 83:1866–1872.[Abstract/Free Full Text]
  10. Arai M, Alpert N.R, MacLennan D.H, Barton P, Periasamy M. Alterations in sarcoplasmic reticulum gene expression in human heart failure. A possible mechanism for alterations in systolic and diastolic properties of the failing myocardium. Circ. Res. (1993) 2:463–469.
  11. Linck B, Boknik P, Eschenhagen T, et al. Messenger RNA expression and immunological quantification of phospholamban and SR-Ca(2+)-ATPase in failing and nonfailing human hearts. Cardiovasc. Res. (1996) 31:625–632.[CrossRef][Web of Science][Medline]
  12. Schwinger R.H, Bohm M, Schmidt U, et al. Unchanged protein levels of SERCA II and phospholamban but reduced Ca2+ uptake and Ca(2+)-ATPase activity of cardiac sarcoplasmic reticulum from dilated cardiomyopathy patients compared with patients with nonfailing hearts. Circulation (1995) 92:3220–3228.[Abstract/Free Full Text]
  13. Dash R, Frank K, Carr A.N, Moravec C.S, Kranias E.G. Gender influences on sarcoplasmic reticulum Ca2+-handling in failing human myocardium. J. Mol. Cell. Cardiol. (2001) 33:1345–1353.[CrossRef][Web of Science][Medline]
  14. Marx S.O, Reiken S, Hisamatsu Y, et al. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell (2000) 101:365–376.[CrossRef][Web of Science][Medline]
  15. Luo W, Grupp I.L, Harrer J, et al. Targeted ablation of the phospholamban gene is associated with markedly enhanced myocardial contractility and loss of beta-agonist stimulation. Circ. Res. (1994) 75:401–409.[Abstract/Free Full Text]
  16. Lu W, Wolska B.M, Grupp I.L, et al. Phospholamban gene dosage effects in the mammalian heart. Circ. Res. (1996) 78:839–847.[Abstract/Free Full Text]
  17. del Monte F, Harding S.E, Schmid U, et al. Restoration of contractile function in isolated cardiomyocytes from failing human hearts by gene transfer of SERCA2a. Circulation (1999) 100:2308–2311.[Abstract/Free Full Text]
  18. He H, Meyer M, Martin J.L, et al. Effects of mutant and antisense RNA of phospholamban on SR Ca(2+)-ATPase activity and cardiac myocyte contractility. Circulation (1999) 100:974–980.[Abstract/Free Full Text]
  19. Schmidt A.G, Edes I, Kranias E.G. Phospholamban: a promising therapeutic target in heart failure? Cardiovasc. Drugs Ther. (2001) 15:387–396.[CrossRef][Web of Science][Medline]
  20. Kubota T, McTiernan C.F, Frye C.S, et al. Dilated cardiomyopathy in transgenic mice with cardiac-specific overexpression of tumor necrosis factor-alpha. Circ. Res. (1997) 81:627–635.[Abstract/Free Full Text]
  21. Kadokami T, McTiernan C.F, Kubota T, Frye C.S, Feldman A.M. Sex-related survival differences in murine cardiomyopathy are associated with differences in TNF-receptor expression. J. Clin. Invest. (2000) 106:589–597.[Web of Science][Medline]
  22. Li Y.Y, Feng Y.Q, Kadokami T, et al. Myocardial extracellular matrix remodeling in transgenic mice overexpressing tumor necrosis factor alpha can be modulated by anti-tumor necrosis factor alpha therapy. Proc. Natl. Acad. Sci. U. S. A. (2000) 97:12746–12751.[Abstract/Free Full Text]
  23. London B, Baker L.C, Lee J.S, et al. Calcium-dependent arrhythmias in transgenic mice with heart failure. Am. J. Physiol. Heart Circ. Physiol. (2003) 284:H431–H441.[Abstract/Free Full Text]
  24. Janczewski A.M, Kadokami T, Lemster B.H, Frye C.S, McTiernan C.F, Feldman A.M. Morphological and functional changes in cardiac myocytes isolated from mice overexpressing TNF-alpha. Am. J. Physiol. Heart Circ. Physiol. (2003) 284:H960–H969.[Abstract/Free Full Text]
  25. Wolska B.M, Stojanovic M.O, Luo W, Kranias E.G, Solaro R.J. Effect of ablation of phospholamban on dynamics of cardiac myocyte contraction and intracellular Ca2+. Am. J. Physiol. (1996) 271:C391–C397.[Web of Science][Medline]
  26. Hoit B.D, Khoury S.F, Kranias E.G, Ball N, Walsh R.A. In vivo echocardiographic detection of enhanced left ventricular function in gene-targeted mice with phospholamban deficiency. Circ. Res. (1995) 77:632–637.[Abstract/Free Full Text]
  27. Severs N.J. Pathophysiology of gap junctions in heart disease. J. Cardiovasc. Electrophysiol. (1994) 5:462–475.[Web of Science][Medline]
  28. Sadoshima J, Xu Y, Slayter H.S, Izumo S. Autocrine release of angiotensin II mediates stretch-induced hypertrophy of cardiac myocytes in vitro. Cell (1993) 5:977–984.
  29. Zhang T, Johnson E.N, Gu Y, et al. The cardiac-specific nuclear delta(B) isoform of Ca2+/calmodulin-dependent protein kinase II induces hypertrophy and dilated cardiomyopathy associated with increased protein phosphatase 2A activity. J. Biol. Chem. (2002) 277:1261–1267.[Abstract/Free Full Text]
  30. Molkentin J.D, Lu J.R, Antos C.L, et al. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell (1998) 93:215–228.[CrossRef][Web of Science][Medline]
  31. Koss K.L, Kranias E.G. Phospholamban: a prominent regulator of myocardial contractility. Circ. Res. (1996) 79:1059–1063.[Free Full Text]
  32. Brittsan A.G, Kranias E.G. Phospholamban and cardiac contractile function. J. Mol. Cell. Cardiol. (2000) 32:2131–2139.[CrossRef][Web of Science][Medline]
  33. Sussman M.A, Welch S, Gude N, et al. Pathogenesis of dilated cardiomyopathy: molecular, structural, and population analyses in tropomodulin-overexpressing transgenic mice. Am. J. Pathol. (1999) 155:2101–2113.[Abstract/Free Full Text]
  34. Freeman K, Lerman I, Kranias E.G, et al. Alterations in cardiac adrenergic signaling and calcium cycling differentially affect the progression of cardiomyopathy. J. Clin. Invest. (2001) 107:967–974.[Web of Science][Medline]
  35. Song Q, Schmidt A.G, Hahn H.S, et al. Rescue of cardiomyocyte dysfunction by phospholamban ablation does not prevent ventricular failure in genetic hypertrophy. J. Clin. Invest. (2003) 111:859–867.[CrossRef][Web of Science][Medline]
  36. Sato Y, Ferguson D.G, Sako H, et al. Cardiac specific overexpression of mouse cardiac calsequestrin is associated with hypertrophy and altered cardiovascular function in transgenic mice. J. Biol. Chem. (1998) 43:28470–28477.
  37. Minamisawa S, Hoshijima M, Chu G, et al. Chronic phospholamban-sarcoplasmic reticulum calcium ATPase interaction is the critical calcium cycling defect in dilated cardiomyopathy. Cell (1999) 99:313–322.[CrossRef][Web of Science][Medline]
  38. Feldman A.M, Combes A, Wagner D, et al. The role of tumor necrosis factor in the pathophysiology of heart failure. J. Am. Coll. Cardiol. (2000) 35:537–544.[Abstract/Free Full Text]
  39. Sato Y, Kiriazis H, Yatani A, et al. Rescue of contractile parameters and myocyte hypertrophy in calsequestrin overexpressing myocardium by phospholamban ablation. J. Biol. Chem. (2001) 276:9392–9399.[Abstract/Free Full Text]
  40. Delling U, Sussman M.A, Molkentin J.D. Re-evaluating sarcoplasmic reticulum function in heart failure. Nat. Med. (2000) 6:942–943.[Web of Science][Medline]
  41. Desai K.H, Schauble E, Luo W, Kranias E, Bernstein D. Phospholamban deficiency does not compromise exercise capacity. Am. J. Physiol. (1997) 276:H1172–H1177.
  42. Force T, Michael A, Kilter H, Haq S. Stretch-activated pathways and left ventricular remodeling. J. Card. Fail. (2002) 8(Suppl. 6):S351–S358.[CrossRef][Web of Science][Medline]
  43. Dolmetsch R.E, Lewis R.S, Goodnow C.C, Healy J.I. Differential activation of transcription factors induced by Ca2+ response amplitude and duration. Nature (1997) 386:855–858.[CrossRef][Medline]
  44. Chu G, Luo W, Slack J.P, et al. Compensatory mechanisms associated with the hyperdynamic function of phospholamban-deficient mouse hearts. Circ. Res. (1996) 79:1064–1076.[Abstract/Free Full Text]
  45. Cross H.R, Kranias E.G, Murphy E, Steenbergen C. Ablation of PLB exacerbates ischemic injury to a lesser extent in female than male mice: protective role of NO. Am. J. Physiol. (2003) 284:H683–H690.[Web of Science]
  46. Cain B.S, Harken A.H, Meldrum D.R. Therapeutic strategies to reduce TNF-alpha mediated cardiac contractile depression following ischemia and reperfusion. J. Mol. Cell. Cardiol. (1999) 31:931–947.[CrossRef][Web of Science][Medline]
  47. Haghighi K, Kolokathis F, Pater L, et al. Human phospholamban null results in lethal dilated cardiomyopathy revealing a critical difference between mouse and human. J. Clin. Invest. (2003) 111:869–876.[CrossRef][Web of Science][Medline]
  48. Kass D.A, Hare J.M, Georgakopoulos D. Murine cardiac function: a cautionary tail. Circ. Res. (1998) 8:519–522.
  49. Haghighi K, Schmidt A.G, Hoit B.D, et al. Superinhibition of sarcoplasmic reticulum function by phospholamban induces cardiac contractile failure. J. Biol. Chem. (2000) 276:24145–24152.[CrossRef][Web of Science]
  50. Weinberg E.O, Thienelt C.D, Katz S.E, et al. Gender differences in molecular remodeling in pressure overload hypertrophy. J. Am. Coll. Cardiol. (1999) 34:264–273.[Abstract/Free Full Text]
  51. Leri A, Malhotra A, Liew C.C, Kajstura J, Anversa P. Telomerase activity in rat cardiac myocytes is age and gender dependent. J. Mol. Cell. Cardiol. (2000) 32:385–390.[CrossRef][Web of Science][Medline]
  52. Dash R, Schmidt A.G, Pathak A, et al. Differential regulation of p38 mitogen-activated protein kinase mediates gender-dependent catecholamine-induced hypertrophy. Cardiovasc. Res. (2003) 57:704–714.[Abstract/Free Full Text]
  53. Malhotra, Buttrick P, Scheuer J. Effects of sex hormones on development of physiological and pathological cardiac hypertrophy in male and female rats. Am. J. Physiol. (1990) 259:H866–H871.[Web of Science][Medline]
  54. Yano M, Kohno M, Ohkusa T, et al. Effect of milrinone on left ventricular relaxation and Ca(2+) uptake function of cardiac sarcoplasmic reticulum. Am. J. Physiol. (2000) 279:H1898–H1905.[Web of Science]
  55. Colucci W.S, Sonnenblick E.H, Adams K.F, et al. Efficacy of phosphodiesterase inhibition with milrinone in combination with converting enzyme inhibitors in patients with heart failure. The Milrinone Multicenter Trials Investigators. J. Am. Coll. Cardiol. (1993) 22:113A–118A.[Medline]
  56. Chu G, Carr A.N, Young K.B, et al. Enhanced myocyte contractility and Ca2+ handling in a calcineurin transgenic model of heart failure. Cardiovasc. Res. (2002) 54:105–116.[Abstract/Free Full Text]
  57. Hoshijima M, Ikeda Y, Iwanaga Y, et al. Chronic suppression of heart-failure progression by a pseudophosphorylated mutant of phospholamban via in vivo cardiac rAAV gene delivery. Nat. Med. (2002) 8:864–871.[Web of Science][Medline]
  58. Gao Y, Levine B.A, Mornet D, Slatter D.A, Strasburg G.M. Interaction of calmodulin with phospholamban and caldesmon: comparative studies by 1H-NMR spectroscopy. Biochim. Biophys. Acta (1992) 1160:22–34.[CrossRef][Medline]
  59. Asahi M, Kurzydlowski K, Tada M, MacLennan D.H. Sarcolipin inhibits polymerization of phospholamban to induce superinhibition of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs). J. Biol. Chem. (2002) 277:26725–26728.[Abstract/Free Full Text]
  60. Koller A, Schlossmann J, Ashman K, Uttenweiler-Joseph S, Ruth P, Hofmann F. Association of phospholamban with a cGMP kinase signaling complex. Biochem. Biophys. Res. Commun. (2003) 300:155–160.[CrossRef][Web of Science][Medline]
  61. Jalil J.E, Doering C.W, Janicki J.S, Pick R, Shroff S.G, Weber K.T. Fibrillar collagen and myocardial stiffness in the intact hypertrophied rat left ventricle. Circ. Res. (1989) 64:1041–1050.[Abstract/Free Full Text]
  62. McLenachan J.M, Dargie H.J. Ventricular arrhythmias in hypertensive left ventricular hypertrophy. Relationship to coronary artery disease, left ventricular dysfunction, and myocardial fibrosis. Am. J. Hypertens. (1990) 3:735–740.[Web of Science][Medline]
  63. Spach M.S, Boineau J.P. Microfibrosis produces electrical load variations due to loss of side-to-side cell connections: a major mechanism of structural heart disease arrhythmias. Pacing Clin. Electrophysiol. (1997) 20:397–413.[CrossRef][Medline]
  64. Dupont E, Matsushita T, Kaba R.A, et al. Altered connexin expression in human congestive heart failure. J. Mol. Cell. Cardiol. (2001) 33:359–371.[CrossRef][Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. R. Waggoner, K. S. Ginsburg, B. Mitton, K. Haghighi, J. Robbins, D. M. Bers, and E. G. Kranias
Phospholamban overexpression in rabbit ventricular myocytes does not alter sarcoplasmic reticulum Ca transport
Am J Physiol Heart Circ Physiol, March 1, 2009; 296(3): H698 - H703.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
L. E. Vinge, P. W. Raake, and W. J. Koch
Gene Therapy in Heart Failure
Circ. Res., June 20, 2008; 102(12): 1458 - 1470.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
S. M. Day, P. Coutu, W. Wang, T. Herron, I. Turner, M. Shillingford, N. C. LaCross, K. L. Converso, L. Piao, J. Li, et al.
Cardiac-directed parvalbumin transgene expression in mice shows marked heart rate dependence of delayed Ca2+ buffering action
Physiol Genomics, May 1, 2008; 33(3): 312 - 322.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
X. Yan, A. J. T. Schuldt, R. L. Price, I. Amende, F.-F. Liu, K. Okoshi, K. K. L. Ho, A. J. Pope, T. K. Borg, B. H. Lorell, et al.
Pressure overload-induced hypertrophy in transgenic mice selectively overexpressing AT2 receptors in ventricular myocytes
Am J Physiol Heart Circ Physiol, March 1, 2008; 294(3): H1274 - H1281.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
B. M. R. Carvalho, R. A. Bassani, K. G. Franchini, and J. W. M. Bassani
Enhanced calcium mobilization in rat ventricular myocytes during the onset of pressure overload-induced hypertrophy
Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1803 - H1813.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
J. W.M. Bassani and R. A. Bassani
SERCA upregulation: Breaking the positive feedback in heart failure?
Cardiovasc Res, September 1, 2005; 67(4): 581 - 582.
[Full Text] [PDF]

Home page
 Circ. Res.Home page
H. C. Champion
Targeting Protein Phosphatase 1 in Heart Failure
Circ. Res., April 15, 2005; 96(7): 708 - 710.
[Full Text] [PDF]

Home page
 Cardiovasc ResHome page
A.-S. Armand and L. J De Windt
Calcium cycling in heart failure: how the fast became too furious
Cardiovasc Res, June 1, 2004; 62(3): 439 - 441.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Janczewski, A. M
Right arrow Articles by McTiernan, C. F
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Janczewski, A. M
Right arrow Articles by McTiernan, C. F
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?